Compositions and Methods for Treating Cancer with Arginine Depletion and Immuno Oncology Agents

ABSTRACT

Methods of treating tumors or cancer include administration of an arginine depleting enzyme and an immune-oncology agent.

BACKGROUND OF THE INVENTION

It has been recognized for over 50 years that certain tumor cells have ahigh demand for amino acids, such as L-arginine and are killed underconditions of L-arginine depletion (Wheatley and Campbell, 2002). Inhuman cells L-arginine is synthesized in three steps; first L-citrullineis synthesized from L-ornithine and carbamoyl phosphate by the enzymeornithine transcarbamylase (OTC), argininosuccinate synthetase (ASS)converts L-citrulline and aspartate to argininosuccinate, followed byconversion of argininosuccinate to L-arginine and fumarate byargininosuccinate lyase (ASL). A large number of hepatocellularcarcinomas (HCC), melanomas and, renal cell carcinomas (Ensor et al.,2002; Feun et al., 2007; Yoon et al., 2007) do not express ASS and thusare sensitive to L-arginine depletion. The molecular basis for the lackof ASS expression appears to be diverse and includes aberrant generegulation. Whereas non-malignant cells enter into quiescence (G₀) whendepleted of L-arginine and thus remain viable for several weeks, tumorcells have cell cycle defects that lead to the re-initiation of DNAsynthesis even though protein synthesis is inhibited, in turn resultingin major imbalances and rapid cell death (Shen et al., 2006; Scott etal., 2000). The selective toxicity of L-arginine depletion for HCC,melanoma and other ASS-deficient cancer cells has been extensivelydemonstrated in vitro, in xenograft animal models and in clinical trials(Ensor et al., 2002; Feun et al., 2007; Shen et al., 2006; Izzo et al.,2004). Recently Cheng et al. (2007) demonstrated that many HCC cells arealso deficient in ornithine transcarbamylase expression and thus, theyare also susceptible to enzymatic L-arginine depletion.

There is interest in the use of L-arginine hydrolytic enzymes for cancertherapy, especially the treatment of cancers such as hepatocarcinomas,melanomas and renal cell carcinomas, for example, which are common formsof cancer associated with high morbidity. Two L-arginine degradingenzymes have been used for cancer therapy: bacterial arginine deiminaseand human arginases. Unfortunately, both of these enzymes displaysignificant shortcomings that present major impediments to clinical use(immunogenicity, and low catalytic activity with very poor stability inserum, respectively). Thus, the therapeutic success of L-argininedepletion therapy will rely on addressing these shortcomings.

Another challenge in the treatment of many cancers is the ability ofsome cancers to evade the immune system. Some tumors, for example, dothis through the immune checkpoint pathways, which are inhibitorypathways in the immune system that maintain self-tolerance by modulatingimmune response. These pathways can be dysregulated by tumors resultingin immune resistance. Some of these pathways, both agonists ofprostimulatory receptors or antagonists of inhibitory signals, both ofwhich result in amplification of antigen-specific T-cell responses, havebecome targets for cancer immunotherapy. Some exemplary receptors andligands include cytotoxic T-lymphocyte-associated antigen 4 (CTLA4),programmed cell death 1 (PD1), programmed cell death ligand 1 (PDL1),lymphocyte activation gene 3 (LAG3), B7-H3, B-7-H4, and T cell membraneprotein 3 (TIM3) among others. (Pardoll, 2012).

SUMMARY OF THE INVENTION

An aspect of the present disclosure generally relates to compositionsand methods for the treatment of cancer with enzymes that depleteL-arginine in serum. In some embodiments, the cancer is one that doesnot express, or is otherwise deficient in, argininosuccinate synthetase(ASS), ornithine transcarbamylase (OTC), or argininosuccinate lyase(ASL).

In some aspects, the present invention also contemplates the use ofarginase proteins wherein the natural metal cofactor (Mn²⁺) is replacedwith another metal. In particular embodiments, the arginase proteincomprises an amino acid sequence of human Arginase I or an amino acidsequence of human Arginase II and a non-native metal cofactor. In someembodiments, the metal is cobalt (Co²⁺). Human Arginase I and IIproteins of the present invention have two Mn (II) sites; either or bothsites can be substituted so as to generate a modified Arginase I or IIprotein with a non-native metal cofactor. In some embodiments, theprotein displays a k_(cat)/K_(M) greater than 400 mM⁻¹ s⁻¹ at pH 7.4. Ina particular embodiment, the protein displays a k_(cat)/K_(M) between400 mM⁻¹ s⁻¹ and 4,000 mM⁻¹ s⁻¹ at pH 7.4. In another embodiment, theprotein displays a k_(cat)/K_(M) between 400 mM⁻¹ s⁻¹ and 2,500 mM⁻¹ s⁻¹at pH 7.4 at 37° C. In a particular embodiment, the present inventioncontemplates a protein comprising an amino acid sequence of humanArginase I or II and a non-native metal cofactor, wherein said proteinexhibits a k_(cat)/K_(M) greater than 400 mM⁻¹ s⁻¹ at 37° C., pH 7.4.

Yet another aspect of the present disclosure is methods of treatingcancer or tumors by arginine depletion in conjunction with animmunotherapeutic treatment targeting an immune checkpoint pathway, forexample. arginine depletion can be accomplished with administration of ahuman Arginase I or Arginase II enzyme, including engineered orderivatized arginase enzymes as well as arginase or other argininedepleting enzymes from other species that exhibit at least an additiveor synergistic effect when administered with an immune checkpointtargeted therapy.

The present disclosure can be described in certain embodiments,therefore, as a method of inhibiting tumor growth in a subject,comprising administering a pharmaceutical composition including atherapeutic amount of a human Arginase I enzyme comprising a cobaltcofactor and an immuno-oncology agent. The tumor can be of various typesthat respond to arginine depletion therapy and in certain embodiments isan arginine auxotrophic tumor, or includes arginine dependent orauxotrophic cells. In certain embodiments the auxotrophic cells exhibita reduced or inhibited expression of one or more of ASS, OTC, ASL, or acombination thereof, thus requiring the tumor cell to utilize argininefrom the serum.

In certain embodiments the human Arginase I or other enzyme isstabilized by association with a stabilizing agent in order to increasethe half-life of the enzyme in the serum of a patient. As used herein“association” can include any of a number of types of associationincluding, but not limited to covalent or non-covalent bonds, and canalso include a protein fusion expressed from an engineered nucleic acidconstruct, from a hydrogen bonding or hydrophobic interaction and othersknown to those of skill in the art. Stabilizing agents for use in thedisclosed methods can include but are not limited to polyethyleneglycol, often referred to as pegylation, conjugation to one or morehomogenous synthetic protein polymers, referred to as extenylation andcommercially available under the trade name Xten®, conjugation to one ormore Fc fragments or to a serum protein like albumin, for example. Allsuch stabilized enzymes and others that would occur to those of skill inthis art are contemplated by the present disclosure.

The disclosed methods are applicable to both human and non-human animalsubjects including but not limited to veterinary, agricultural, domesticor research animals. It is an aspect of the disclosure that theimmuno-oncology agent enhances the subject's immune response. In certainembodiments enhancing an immune system includes increasing activity of apatient's T-cell response to the presence of a tumor. In certainembodiments, therefore, the immuno-oncology agent inhibits an immunesuppressor, which is sometimes a cell surface receptor referred to as acheckpoint inhibitor, or a ligand for such a receptor. Examples include,but are not limited to PD-1 pathway inhibitors such as an anti-PD-1antibody or an anti-PD-L1 antibody, OX40 (CD134) pathway inhibitors suchas anti-OX40 or anti-OX40(CD252), anti-4-1BB or other anti B7 familyligands such as anti-B7-H1 and anti-B7.1 for example. Exemplaryantibodies include but are not limited to pembrolizumab, ipilimumab,atezolizumab or nivolumab.

The methods of the disclosure are contemplated for the treatment of anyresponsive cancer or tumor, including, but not limited to hepatocellularcarcinoma, renal cell carcinoma, breast cancer, melanoma, prostatecancer, pancreatic cancer, bladder cancer, colon carcinoma, colorectalcancer, triple negative breast cancer, Hodgkin's lymphoma, gastriccancer, glioblastoma, Merkel cell carcinoma, lung carcinoma, small celllung cancers or non-small cell lung cancers. The administration of acombination of the human Arginase I enzyme and the anti-PD-1 antibody oranti-PDL-1 antibody or other immune checkpoint or TNF receptorinhibitors can exhibit an additive effect on tumor growth inhibitioncompared to the tumor growth inhibition exhibited by administering atherapeutic dose of the anti-PD-1 antibody alone or the anti-PD-Liantibody alone, or the human Arginase I enzyme alone, or in certainembodiments exhibits a greater than additive, or synergistic effect onthe tumor growth or cancer. The two treatment regimens can beadministered concurrently or they can be administered sequentially asneeded.

The current disclosure can also be described in certain embodiments as amethod of treating cancer in a cancer patient comprising administeringto said patient a therapeutic amount of a pharmaceutical compositioncomprising a pegylated human Arginase I enzyme comprising a cobaltcofactor and an immune system modulating therapy comprisingadministering a pharmaceutical composition comprising an immuno-oncologyagent.

In certain embodiments a therapeutic amount of the pegylated humanArginase I enzyme comprising a cobalt cofactor is from about 0.01 mg/kgto about 7.5 mg/kg, about 0.05 mg/kg to about 5 mg/kg, or about 0.1mg/kg to about 5 mg/kg, or any amount derivable from or contained withinthe preceding ranges.

The pharmaceutical composition including a pegylated human Arginase Ienzyme comprising a cobalt cofactor can be administered parenterally, orit can be delivered by various routes known in the art, including butnot limited to topically, subcutaneously, intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intraocularly, intranasally, intravitreally, intravaginally,intrarectally, intramuscularly, subcutaneously, subconjunctivally,intravesicularlly, mucosally, intrapericardially, intraumbilically,orally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, or via a lavage. In certain embodiments the pharmaceuticalcomposition is administered intravenously or subcutaneously.

The disclosure can also be described as a method of treating cancer in acancer patient comprising administering to said patient an argininedepleting agent and a checkpoint pathway inhibitor or other immunesystem modulator that inhibits or reduces cancer growth orproliferation. The methods further include treatment of cancers in whichthe therapeutic effect of treatment with the arginine depleting agentand a checkpoint pathway inhibitor is additive as compared to treatmentthe arginine depleting agent alone or said checkpoint pathway inhibitoralone, or in which the therapeutic effect of treatment with saidarginine depleting agent and a checkpoint pathway inhibitor issynergistic as compared to treatment the arginine depleting agent aloneor said checkpoint pathway inhibitor alone. In certain embodiments thetreatment can result in from 50% to 99%, or from 90% to 99% reduction inserum arginine in the patient, or reduction of serum arginine in apatient to an undetectable level.

Enzymes useful in the practice of the methods can include arginaseenzymes, arginine deiminase enzymes or a combination thereof. Theenzymes can be human enzymes, recombinant human enzymes, engineeredhuman enzymes or enzymes from other species, either mammalian orbacterial, for example, including but not limited to mycoplasma.

In some embodiments, the native arginase is modified only by thesubstitution of the metal cofactor. In other embodiments, the arginaseis modified by substitution of the metal cofactor in addition to othermodifications, such as substitutions, deletions, truncations, orstabilization by conjugation to a stabilizing protein or polymer, suchas by pegylation. In a particular embodiment, the invention provides aprotein comprising a native amino acid sequence of human Arginase I orII and a non-native metal cofactor, wherein the amino acid sequence islacking part of the native sequence. In particular embodiments, thenon-native metal cofactor is cobalt. In some embodiments, the arginaselacks a portion of the wild-type sequence. In other embodiments, theamino acid sequence comprises a truncated Arginase I or Arginase IIsequence. In a particular embodiment, the arginase is Arginase II andlacks the first 21 amino acids of the wild-type sequence. In anotherembodiment, the native arginases lacks an N-terminal methionine.

In another aspect, the present invention contemplates an arginaseprotein comprising at least one amino acid substitution, wherein theprotein displays an increased catalytic activity under physiologicalconditions and especially at the pH of human serum (pH 7.4) whencompared with native human Arginase I or II protein. In someembodiments, the arginase protein is a human Arginase I protein or humanArginase II protein. In some embodiments, the protein further comprisesa non-native metal cofactor. In particular embodiments, the non-nativemetal cofactor is Co⁺². Substitution of the Mn⁺² cofactor with Co⁺²results in marked increase in catalytic activity and a drastic reductionin K_(M) at physiological pH. In some aspects, the present inventionalso contemplates fusion proteins comprising an arginase linked to anon-arginase amino acid sequence. In one embodiment, the non-arginasesequence comprises at least a portion of the Fc region of animmunoglobulin, e.g., to increase the half-life of the arginase in serumwhen administered to a patient. The Fc region or portion thereof may beany suitable Fc region. In one embodiment, the Fc region or portionthereof is an IgG Fc region. In some embodiments, the amino acidsequence having arginase activity is selected from the group consistingof a native or mutated amino acid sequence of human Arginase I and anative or mutated amino acid sequence of human Arginase II or otherarginine depleting enzymes known in the art. In certain embodiments, adimeric Fc-arginase fusion protein, albumin, or a synthetic proteinconjugation is contemplated.

The arginase in the fusion protein may be native, mutated, and/orotherwise modified, e.g., metal cofactor modified. In some embodiments,the arginase may contain deletions, substitutions, truncations or acombination thereof. In a particular embodiment, the present inventioncontemplates an Fc-arginase containing fusion protein, wherein thearginase is an Arginase I. In one embodiment, the arginase lacks aportion of the wild-type sequence. In another embodiment, the arginaseis Arginase I lacking an N-terminal methionine. In yet anotherembodiment, the arginase is Arginase II, wherein the Arginase II lacksthe first 21 amino acids of the wild-type Arginase II sequence. In someembodiments, the arginase further comprises a non-native metal cofactor.In these embodiments, either or both sites can be substituted togenerate a fusion protein comprising an amino acid sequence of humanArginase I or II and a non-native metal cofactor. In some embodiments,the non-native metal cofactor is cobalt. In some embodiments, thearginase contains a substitution. Exemplary arginase enzymes for use inthe present disclosure are more fully described in U.S. Pat. No.8,440,184, incorporated herein in its entirety by reference.

The present invention also contemplates methods of treatment by theadministration of the arginase proteins of the present invention, and inparticular methods of treating subjects with cancer. In someembodiments, the cancer is one that does not express, or is otherwisedeficient in, ASS, OTC, or ASL. In particular embodiments, the humancancer is an arginine auxotrophic cancer. As discussed above, thearginase protein may be native, mutated, and/or otherwise modified,e.g., metal cofactor modified. In one embodiment, the present inventioncontemplates a method of treating a human cancer patient comprisingadministering a formulation comprising a fusion protein, the fusionprotein comprising an amino acid sequence having arginase activity andat least a portion of the Fc region of a human immunoglobulin to thepatient. In some embodiments, the administration occurs under conditionssuch that at least a portion of the cancer cells of the cancer arekilled. In another embodiment, the formulation comprises an amino acidsequence having human arginase activity higher than that displayed bythe authentic human arginases at physiological conditions and furthercomprising one or more attached polyethylene glycol chain(s). In someembodiment, the formulation is a pharmaceutical formulation comprisingany of the above discussed arginase proteins and a pharmaceuticallyacceptable excipients. Such pharmaceutically acceptable excipients arewell known to those having skill in the art. All of the above arginasevariants are contemplated as useful for human therapy.

The cancer may be any type of cancer or tumor type. In some embodiments,the cancer is hepatocellular carcinoma, renal cell carcinoma, melanoma,prostate cancer, or pancreatic cancer. In some embodiments, theformulation is administered topically, intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intraocularly, intranasally, intravitreally, intravaginally,intrarectally, intramuscularly, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,orally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, or via a lavage.

All of the above mentioned arginases, variants and the like arecontemplated in a preferred embodiment as purified or isolated proteins,and preferably monomeric proteins.

The embodiments in the Example section are understood to be embodimentsof the invention that are applicable to all aspects of the invention.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the composition,device or method being employed to determine the value.

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

The term “therapeutically effective” as used herein refers to an amountof an active agent and/or therapeutic composition (such as a therapeuticpolynucleotide and/or therapeutic polypeptide) that is employed inmethods of the present invention to achieve a therapeutic effect, suchas wherein at least one symptom of a condition being treated is at leastameliorated, and/or to the analysis of the processes or materials usedin conjunction with these cells.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 is a graph showing serum L-arginine depletion in the mouse model.Serum L-Arg concentrations of Balb/c mice treated with a single IP doseof Co-hArgl are kept≤to 3-4 μM for over 3 days.

FIG. 2 is a graph showing HCC tumor xenograft reduction when treatedwith Co-hArgl as compared to controls. Nude mice bearing a Hep3b tumorxenografts were treated twice by IP injection with either PBS (°) orCo-hArgl (●) at day 9 and at day 12. Tumor shrinkage was observed in themice treated with Co-hArgl whereas PBS treated tumors grew unchecked.

FIG. 3 is a graph showing the effect of cobalt loading on the catalyticactivity of human Arginase I.

FIG. 4 is a graph showing colon carcinoma tumor growth inhibition inCT26 mouse model with Co-hArgl, anti PD-L1 and the combination ofCo-hArgl and anti PD-L1. As seen in the data, the combination of the 2agents has a greater than additive effect on inhibition of tumor growth.

FIG. 5 is a graph showing colon carcinoma tumor growth inhibition in anMC38 mouse model with Co-hArgl, anti OX40 antibodies, and thecombination of Co-hArgl and anti OX40 antibodies. As seen in the data,the combination of the 2 agents has a greater than additive effect oninhibition of tumor growth.

FIG. 6 is a graph showing CD45+ Tcells present in CT26 mouse model withCo-hArgl, anti PD-L1 and the combination of Co-hArgl and anti PD-L1.

FIG. 7 is a graph showing percent CD8+ cells present in CD45+ Tcells asshown in FIG. 6.

DESCRIPTION OF THE ILLUSTRATIVE EMBODIMENTS

The invention generally relates to compositions and methods for thetreatment of cancer with enzymes that deplete L-arginine in serum. Insome embodiments, the cancer is one that does not express, or isotherwise deficient in, argininosuccinate synthetase (ASS), ornithinetranscarbamylase (OTC), or argininosuccinate lyase (ASL), or otherenzymes required for arginine biosynthesis. Both native and mutatedenzymes are contemplated, as well as enzymes with modified metalcofactors, enzymes fused to other polypeptides as well as enzymesconjugated to polymers that increase serum persistence, e.g., highmolecular weight polyethylene glycol I. Arginase

Arginase is a manganese-containing enzyme. It is the final enzyme of theurea cycle. Arginase is the fifth and final step in the urea cycle, aseries of biophysical reactions in mammals during which the bodydisposes of harmful ammonia. Specifically, arginases convert L-arginineinto L-ornithine and urea.

L-arginine is the nitrogen donating substrate for nitric oxide synthase(NOS), producing L-citrulline and NO. Although the K_(M) of arginase(2-5 mM) has been reported to be much higher than that of NOS forL-arginine (2-20 μM), arginase may also play a role in regulating NOSactivity. Under certain conditions Arginase I is Cys-S-nitrosylated,resulting in higher affinity for L-arginine and reduced availability ofsubstrate for NOS.

Arginase is a homo-trimeric enzyme with an αβ fold of a paralleleight-stranded βsheet surrounded by several helices. The enzyme containsa di-nuclear metal cluster that is integral to generating a hydroxidefor nucleophilic attack on the guanidinium carbon of L-arginine. Thenative metal for arginase is Mn²⁺. These Mn²⁺ ions coordinate water,orientating and stabilizing the molecule and allowing water to act as anucleophile and attack L-arginine, hydrolyzing it into ornithine andurea.

Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze thehydrolysis of L-arginine to urea and L-ornithine. The Arginase I gene islocated on chromosome 6 (6q.23), is highly expressed in the cytosol ofhepatocytes, and functions in nitrogen removal as the final step of theurea cycle. The Arginase II gene is found on chromosome 14 (14q.24.1).Arginase II is mitochondrially located in tissues such as kidney, brain,and skeletal muscle where it is thought to provide a supply ofL-ornithine for proline and polyamine biosynthesis (Lopez et al., 2005).

Arginases have been investigated for nearly 50 years as a method fordegrading extracellular L-arginine (Dillon et al., 2002). Some promisingclinical results have been achieved by introducing arginase bytranshepatic arterial embolisation; following which, several patientsexperienced partial remission of HCC (Cheng et al., 2005). However,since arginase has a high K_(M) (˜2-5 mM) and exhibits very low activityat physiological pH values, high dosing is required for chemotherapeuticpurposes (Dillon et al., 2002). While native arginase is cleared fromcirculation within minutes (Savoca et al., 1984), a single injection ofPEG-arginase MW5000 in rats was sufficient to achieve near completearginine depletion for ˜3 days (Cheng et al., 2007).

Cheng et al. made the surprising observation that many human HCC cellslines do not express OTC (in addition to ASS) and thus they aresusceptible to PEG-arginase (Cheng et al., 2007). In mice implanted withHep3b hepatocarcinoma cells weekly administration of PEG-arginaseresulted in tumor growth retardation which was accentuated byco-administration of 5-fluorouracil (5-FU). However, PEG-arginase wasused at the very high doses that are impractical for human therapy,reflecting its lower physiological activity.

To address these issues a bacterial arginine hydrolyzing enzyme,arginine deiminase or ADI which displays good kinetics and stability hasbeen tested in vitro and clinically. Unfortunately ADI is a bacterialenzyme and therefore it induces strong immune responses and adverseeffects in most patients. However, for those patients who do not developsignificant adverse responses, an impressive percentage exhibit stabledisease or remission.

For clinical use, it is essential that the arginase is engineered toallow it to persist for long times (e.g., days) in circulation. In theabsence of any modification, human arginase has a half-life of only afew minutes in circulation primarily because its size is notsufficiently large to avoid filtration though the kidneys. Unmodifiedhuman arginase is very susceptible to deactivation in serum and it isdegraded with a half-life of only four hours. Therefore, the presentinvention developed novel and improved forms of arginase for clinicalresearch and potential therapeutic use with improved circulationpersistence. II. Arginase Variants

Mammals have two arginase isozymes (EC 3.5.3.1) that catalyze thehydrolysis of L-arginine to urea and L-ornithine. The Arginase I gene islocated on chromosome 6 (6q.23), is highly expressed in the cytosol ofhepatocytes, and functions in nitrogen removal as the final step of theurea cycle. The Arginase II gene is found on chromosome 14 (14q.24.1).Arginase II is mitochondrially located in tissues such as kidney, brain,and skeletal muscle where it is thought to provide a supply ofL-ornithine for proline and polyamine biosynthesis (Lopez et al., 2005).L-arginine is the sole substrate for nitric oxide synthase (NOS),producing L-citrulline and NO. Although the K_(M) of arginase (2-5 mM)has been reported to be much higher than that of NOS for L-arginine(2-20 μM), arginase may also play a role in regulating NOS activity(Durante et al., 2007). Under certain conditions Arginase I isCys-S-nitrosylated, resulting in higher affinity for L-arginine andreduced availability of substrate for NOS (Santhanam et al., 2007).Arginase is a homo-trimeric enzyme with an α/β fold of a paralleleight-stranded β-sheet surrounded by several helices. The enzymecontains a di-nuclear metal cluster that is integral to generating ahydroxide for nucleophilic attack on the guanidinium carbon ofL-arginine (Cama et al., 2003; Dowling et al., 2008). The native metalfor arginase is Mn²⁺. arginase with the native metal (i.e. Mn2+)exhibits a pH optimum of 9. At physiological pH the enzyme exhibits morethan a 10-fold lower k_(cat)/K_(M), in the hydrolysis of L-arginine. Thelow catalytic activity displayed by the authentic human arginase withthe native Mn²⁺ enzyme presents a problem for human therapy since itmeans that impractical doses of the enzyme may have to be used toachieve a therapeutically relevant reduction in L-arginine plasmalevels.

In some aspects, the present invention contemplates mutant arginaseswherein the natural metal cofactor (Mn²⁺) is replaced with anothermetal. It has been found that substitution of the metal cofactor inhuman arginase exerts a beneficial effect on the rate of hydrolysis ofL-Arginine and stability under physiological conditions when compared tonative human arginase with the natural metal cofactor. The substitutionof the native metal (Mn²⁺) with other divalent cations can be exploitedto shift the pH optimum of the enzyme to a lower values and thus achievehigh rates of L-arginine hydrolysis under physiological conditions.Human Arginase I and II proteins of the present invention have two Mn(II) sites; therefore, either or both sites can be substituted so as togenerate a mutated Arginase I or II protein with a non-native metalcofactor.

In some embodiments, the metal is cobalt (Co²⁺). Incorporation of Co2+in the place of Mn²⁺ in human Arginase I or human Arginase II results indramatically higher activity at physiological pH. It was found that ahuman Arginase I enzyme containing Co²⁺ (“Co-hArgI”) displayed a 10 foldincrease in k_(cat)/K_(M) in vitro at pH 7.4, which in turn translatedinto a 15 fold increase in HCC cytotoxicity and a 13-fold increase inmelanoma cytotoxity as compared to the human Arginase I which containsMn²⁺ (“Mn-hArgl”). It was also found that a pharmacological preparationof Co-hArgI could clear serum L-Arg for over 3 days in mice with asingle injection. Furthermore, it was found that a pharmacologicalpreparation of Co-hArgl could shrink HCC tumor xenografts in nude micewhereas Mn-hArgl only slowed tumor growth (Ensor et al., 2002).

In certain aspects of the invention, methods and compositions related topegylated arginase are disclosed. Specifically, pegylation of arginaseat an engineered cysteine residue (e.g., substituting the third residueof the N-terminal) may be used to produce a homogenous pegylatedarginase composition. Methods for isolation of pegylated arginase basedon temporary disruption of polymerization are also disclosed.

Pegylation is the process of covalent attachment of poly(ethyleneglycol) polymer chains to another molecule, normally a drug ortherapeutic protein. Pegylation is routinely achieved by incubation of areactive derivative of PEG with the target macromolecule. The covalentattachment of PEG to a drug or therapeutic protein can “mask” the agentfrom the host's immune system (reduced immunogenicity and antigenicity),increase the hydrodynamic size (size in solution) of the agent whichprolongs its circulatory time by reducing renal clearance. Pegylationcan also provide water solubility to hydrophobic drugs and proteins.

The first step in pegylation is the suitable functionalization of thePEG polymer at one or both terminals. PEGs that are activated at eachterminus with the same reactive moiety are known as “homobifunctional”,whereas if the functional groups present are different, then the PEGderivative is referred as “heterobifunctional” or “heterofunctional.”The chemically active or activated derivatives of the PEG polymer areprepared to attach the PEG to the desired molecule.

The choice of the suitable functional group for the PEG derivative isbased on the type of available reactive group on the molecule that willbe coupled to the PEG. For proteins, typical reactive amino acidsinclude lysine, cysteine, histidine, arginine, aspartic acid, glutamicacid, serine, threonine, tyrosine. The N-terminal amino group and theC-terminal carboxylic acid can also be used.

The techniques used to form first generation PEG derivatives aregenerally reacting the PEG polymer with a group that is reactive withhydroxyl groups, typically anhydrides, acid chlorides, chloroformatesand carbonates. In the second generation pegylation chemistry moreefficient functional groups such as aldehyde, esters, amides etc. madeavailable for conjugation.

As applications of pegylation have become more and more advanced andsophisticated, there has been an increase in need for heterobifunctionalPEGs for conjugation. These heterobifunctional PEGs are very useful inlinking two entities, where a hydrophilic, flexible and biocompatiblespacer is needed. Preferred end groups for heterobifunctional PEGs aremaleimide, vinyl sulfones, pyridyl disulfide, amine, carboxylic acidsand NHS esters.

The most common modification agents, or linkers, are based on methoxyPEG (mPEG) molecules. Their activity depends on adding aprotein-modifying group to the alcohol end. In some instancespolyethylene glycol (PEG diol) is used as the precursor molecule. Thediol is subsequently modified at both ends in order to make a hetero- orhomo-dimeric PEG-linked molecule (as shown in the example with PEGbis-vinylsulfone). Proteins are generally PEGylated at nucleophilicsites such as unprotonated thiols (cysteinyl residues) or amino groups.Examples of cysteinyl-specific modification reagents include PEGmaleimide, PEG iodoacetate, PEG thiols, and PEG vinylsulfone. All fourare strongly cysteinyl-specific under mild conditions and neutral toslightly alkaline pH but each has some drawbacks. The amide formed withthe maleimides can be somewhat unstable under alkaline conditions sothere may be some limitation to formulation options with this linker.The amide linkage formed with iodo PEGs is more stable, but free iodinecan modify tyrosine residues under some conditions. PEG thiols formdisulfide bonds with protein thiols, but this linkage can also beunstable under alkaline conditions. PEG-vinylsulfone reactivity isrelatively slow compared to maleimide and iodo PEG; however, thethioether linkage formed is quite stable. Its slower reaction rate alsocan make the PEG-vinylsulfone reaction easier to control.

Site-specific pegylation at native cysteinyl residues is seldom carriedout, since these residues are usually in the form of disulfide bonds orare required for biological activity. On the other hand, site-directedmutagenesis can be used to incorporate cysteinyl pegylation sites forthiol-specific linkers. The cysteine mutation must be designed such thatit is accessible to the pegylation reagent and is still biologicallyactive after pegylation.

Amine-specific modification agents include PEG NHS ester, PEG tresylate,PEG aldehyde, PEG isothiocyanate, and several others. All react undermild conditions and are very specific for amino groups. The PEG NHSester is probably one of the more reactive agents; however, its highreactivity can make the pegylation reaction difficult to control atlarge scale. PEG aldehyde forms an imine with the amino group, which isthen reduced to a secondary amine with sodium cyanoborohydride. Unlikesodium borohydride, sodium cyanoborohydride will not reduce disulfidebonds. However; this chemical is highly toxic and must be handledcautiously, particularly at lower pH where it becomes volatile.

Due to the multiple lysine residues on most proteins, site-specificpegylation can be a challenge. Fortunately, because these reagents reactwith unprotonated amino groups, it is possible to direct the pegylationto lower-pK amino groups by performing the reaction at a lower pH.Generally the pK of the a-amino group is 1-2 pH units lower than theepsilon-amino group of lysine residues. By PEGylating the molecule at pH7 or below, high selectivity for the N-terminus frequently can beattained. However; this is only feasible if the N-terminal portion ofthe protein is not required for biological activity. Still, thepharmacokinetic benefits from pegylation frequently outweigh asignificant loss of in vitro bioactivity, resulting in a product withmuch greater in vivo bioactivity regardless of pegylation chemistry.

There are several parameters to consider when developing a pegylationprocedure. Fortunately, there are usually no more than four or five keyparameters. The “design of experiments” approach to optimization ofpegylation conditions can be very useful. For thiol-specific pegylationreactions, parameters to consider include: protein concentration,PEG-to-protein ratio (on a molar basis), temperature, pH, reaction time,and in some instances, the exclusion of oxygen. Oxygen can contribute tointermolecular disulfide formation by the protein, which will reduce theyield of the PEGylated product. The same factors should be considered(with the exception of oxygen) for amine-specific modification exceptthat pH may be even more critical, particularly when targeting theN-terminal amino group.

For both amine- and thiol-specific modifications, the reactionconditions may affect the stability of the protein. This may limit thetemperature, protein concentration, and pH. In addition, the reactivityof the PEG linker should be known before starting the pegylationreaction. For example, if the pegylation agent is only 70% active, theamount of PEG used should ensure that only active PEG molecules arecounted in the protein-to-PEG reaction stoichiometry. How to determinePEG reactivity and quality will be described later.

IV. Proteins and Peptides

In certain embodiments, the present invention concerns novelcompositions comprising at least one protein or peptide, such asstabilized arginase multimers. These peptides may be comprised in afusion protein or conjugated to an agent as described supra.

A. Proteins and Peptides

As used herein, a protein or peptide generally refers, but is notlimited to, a protein of greater than about 200 amino acids, up to afull length sequence translated from a gene; a polypeptide of greaterthan about 100 amino acids; and/or a peptide of from about 3 to about100 amino acids. For convenience, the terms “protein,” “polypeptide” and“peptide” are used interchangeably herein.

In certain embodiments the size of at least one protein or peptide maycomprise, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about110, about 120, about 130, about 140, about 150, about 160, about 170,about 180, about 190, about 200, about 210, about 220, about 230, about240, about 250, about 275, about 300, about 325, about 350, about 375,about 400, about 425, about 450, about 475, about 500, about 525, about550, about 575, about 600, about 625, about 650, about 675, about 700,about 725, about 750, about 775, about 800, about 825, about 850, about875, about 900, about 925, about 950, about 975, about 1000, about 1100,about 1200, about 1300, about 1400, about 1500, about 1750, about 2000,about 2250, about 2500 or greater amino acid residues.

As used herein, an “amino acid residue” refers to any naturallyoccurring amino acid, any amino acid derivative or any amino acid mimicknown in the art. In certain embodiments, the residues of the protein orpeptide are sequential, without any non-amino acid interrupting thesequence of amino acid residues. In other embodiments, the sequence maycomprise one or more non-amino acid moieties. In particular embodiments,the sequence of residues of the protein or peptide may be interrupted byone or more non-amino acid moieties. Accordingly, the term “protein orpeptide” encompasses amino acid sequences comprising at least one of the20 common amino acids found in naturally occurring proteins, or at leastone modified or unusual amino acid, including but not limited to thoseshown on Table 1 below.

TABLE 1 Modified and Unusual Amino Acids Abbr. Amino Acid Aad2-Aminoadipic acid Baad 3-Aminoadipic acid Bala β-alanine,β-Amino-propionic acid Abu 2-Aminobutyric acid 4Abu 4-Aminobutyric acid,piperidinic acid Acp 6-Aminocaproic acid Ahe 2-Aminoheptanoic acid Aib2-Aminoisobutyric acid Baib 3-Aminoisobutyric acid Apm 2-Aminopimelicacid Dbu 2,4-Diaminobutyric acid Des Desmosine Dpm 2,2′-Diaminopimelicacid Dpr 2,3-Diaminopropionic acid EtGly N-Ethylglycine EtAsnN-Ethylasparagine Hyl Hydroxylysine AHyl allo-Hydroxylysine 3Hyp3-Hydroxyproline 4Hyp 4-Hydroxyproline Ide Isodesmosine Alleallo-Isoleucine MeGly N-Methylglycine, sarcosine MeIleN-Methylisoleucine MeLys 6-N-Methyllysine MeVal N-Methylvaline NvaNorvaline Nle Norleucine Orn Ornithine

Proteins or peptides may be made by any technique known to those ofskill in the art, including the expression of proteins, polypeptides orpeptides through standard molecular biological techniques, the isolationof proteins or peptides from natural sources, or the chemical synthesisof proteins or peptides. The nucleotide and protein, polypeptide andpeptide sequences corresponding, to various genes have been previouslydisclosed, and may be found at computerized databases known to those ofordinary skill in the art. One such database is the National Center forBiotechnology Information's Genbank and GenPept databases (available onthe world wide web at ncbi.nlm.nih.govf). The coding regions for knowngenes may be amplified and/or expressed using the techniques disclosedherein or as would be know to those of ordinary skill in the art.Alternatively, various commercial preparations of proteins, polypeptidesand peptides are known to those of skill in the art.

B. Nucleic Acids and Vectors

In certain aspects of the invention, nucleic acid sequences encoding afusion protein as a stabilized multimeric arginase may be disclosed.Depending on which expression system to be used, nucleic acid sequencescan be selected based on conventional methods. For example, humanarginase I and II contain multiple codons that are rarely utilized in E.coli that may interfere with expression, therefore the respective genesor variants thereof may be codon optimized for E. coli expression.Various vectors may be also used to express the protein of interest,such as a fusion multimeric arginase or a cysteine-substituted arginase.Exemplary vectors include, but are not limited, plasmid vectors, viralvectors, transposon or liposome-based vectors.

C. Host Cells

Host cells, preferably eukaryotic cells, useful in the present inventionare any that may be transformed to allow the expression and secretion ofarginase and fusion multimers thereof. The host cells may be bacteria,mammalian cells, yeast, or filamentous fungi. Various bacteria includeEscherichia and Bacillus. Yeasts belonging to the genera Saccharomyces,Kluyveromyces, Hansenula, or Pichia would find use as an appropriatehost cell. Various species of filamentous fungi may be used asexpression hosts including the following genera: Aspergillus,Trichoderma, Neurospora, Penicillium, Cephalosporium, Achlya, Podospora,Endothia, Mucor, Cochliobolus and Pyricularia.

Examples of usable host organisms include bacteria, e.g., Escherichiacoli MC1061, derivatives of Bacillus subtilis BRB1 (Sibakov et al.,1984), Staphylococcus aureus SAI123 (Lordanescu, 1975) or Streptococcuslividans (Hopwood et al., 1985); yeasts, e.g., Saccharomyces cerevisiaeAH 22 (Mellor et al., 1983) and Schizosaccharomyces pombe; filamentousfungi, e.g., Aspergillus nidulans, Aspergillus awamori (Ward, 1989),Trichoderma reesei (Penttila et al., 1987; Harkki et al, 1989).

Examples of mammalian host cells include Chinese hamster ovary cells(CHO-K1; ATCC CCL61), rat pituitary cells (GH₁; ATCC CCL82), HeLa S3cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E; ATCCCRL 1548)SV40-transformed monkey kidney cells (COS-1; ATCC CRL 1650) and murineembryonic cells (NIH-3T3; ATCC CRL 1658). The foregoing beingillustrative but not limitative of the many possible host organismsknown in the art. In principle, all hosts capable of secretion can beused whether prokaryotic or eukaryotic.

Mammalian host cells expressing the arginase and/or their fusionmultimers are cultured under conditions typically employed to culturethe parental cell line. Generally, cells are cultured in a standardmedium containing physiological salts and nutrients, such as standardRPMI, MEM, IMEM or DMEM, typically supplemented with 5-10% serum, suchas fetal bovine serum. Culture conditions are also standard, e.g.,cultures are incubated at 37° C. in stationary or roller cultures untildesired levels of the proteins are achieved.

D. Protein Purification

Protein purification techniques are well known to those of skill in theart. These techniques involve, at one level, the homogenization andcrude fractionation of the cells, tissue or organ to polypeptide andnon-polypeptide fractions. The protein or polypeptide of interest may befurther purified using chromatographic and electrophoretic techniques toachieve partial or complete purification (or purification tohomogeneity) unless otherwise specified. Analytical methods particularlysuited to the preparation of a pure peptide are ion-exchangechromatography, gel exclusion chromatography, polyacrylamide gelelectrophoresis, affinity chromatography, immunoaffinity chromatographyand isoelectric focusing. A particularly efficient method of purifyingpeptides is fast performance liquid chromatography (FPLC) or even highperformance liquid chromatography (HPLC).

A purified protein or peptide is intended to refer to a composition,isolatable from other components, wherein the protein or peptide ispurified to any degree relative to its naturally-obtainable state. Anisolated or purified protein or peptide, therefore, also refers to aprotein or peptide free from the environment in which it may naturallyoccur. Generally, “purified” will refer to a protein or peptidecomposition that has been subjected to fractionation to remove variousother components, and which composition substantially retains itsexpressed biological activity. Where the term “substantially purified”is used, this designation will refer to a composition in which theprotein or peptide forms the major component of the composition, such asconstituting about 50%, about 60%, about 70%, about 80%, about 90%,about 95%, or more of the proteins in the composition.

Various techniques suitable for use in protein purification are wellknown to those of skill in the art. These include, for example,precipitation with ammonium sulphate, PEG, antibodies and the like, orby heat denaturation, followed by: centrifugation; chromatography stepssuch as ion exchange, gel filtration, reverse phase, hydroxylapatite andaffinity chromatography; isoelectric focusing; gel electrophoresis; andcombinations of these and other techniques. As is generally known in theart, it is believed that the order of conducting the variouspurification steps may be changed, or that certain steps may be omitted,and still result in a suitable method for the preparation of asubstantially purified protein or peptide.

Various methods for quantifying the degree of purification of theprotein or peptide are known to those of skill in the art in light ofthe present disclosure. These include, for example, determining thespecific activity of an active fraction, or assessing the amount ofpolypeptides within a fraction by SDS/PAGE analysis. A preferred methodfor assessing the purity of a fraction is to calculate the specificactivity of the fraction, to compare it to the specific activity of theinitial extract, and to thus calculate the degree of purity therein,assessed by a “-fold purification number.” The actual units used torepresent the amount of activity will, of course, be dependent upon theparticular assay technique chosen to follow the purification, andwhether or not the expressed protein or peptide exhibits a detectableactivity.

There is no general requirement that the protein or peptide always beprovided in their most purified state. Indeed, it is contemplated thatless substantially purified products may have utility in certainembodiments. Partial purification may be accomplished by using fewerpurification steps in combination, or by utilizing different forms ofthe same general purification scheme. For example, it is appreciatedthat a cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “-fold” purification thanthe same technique utilizing a low pressure chromatography system.Methods exhibiting a lower degree of relative purification may haveadvantages in total recovery of protein product, or in maintaining theactivity of an expressed protein.

In certain embodiments a protein or peptide may be isolated or purified,for example, a stabilized arginase multimeric fusion protein, or anarginase prior or post pegylation. For example, a His tag or an affinityepitope may be comprised in such a arginase variant to facilitatepurification. Affinity chromatography is a chromatographic procedurethat relies on the specific affinity between a substance to be isolatedand a molecule to which it can specifically bind. This is areceptor-ligand type of interaction. The column material is synthesizedby covalently coupling one of the binding partners to an insolublematrix. The column material is then able to specifically adsorb thesubstance from the solution. Elution occurs by changing the conditionsto those in which binding will not occur (e.g., altered pH, ionicstrength, temperature, etc.). The matrix should be a substance thatitself does not adsorb molecules to any significant extent and that hasa broad range of chemical, physical and thermal stability. The ligandshould be coupled in such a way as to not affect its binding properties.The ligand should also provide relatively tight binding. And it shouldbe possible to elute the substance without destroying the sample or theligand.

Size exclusion chromatography (SEC) is a chromatographic method in whichmolecules in solution are separated based on their size, or in moretechnical terms, their hydrodynamic volume. It is usually applied tolarge molecules or macromolecular complexes such as proteins andindustrial polymers. Typically, when an aqueous solution is used totransport the sample through the column, the technique is known as gelfiltration chromatography, versus the name gel permeation chromatographywhich is used when an organic solvent is used as a mobile phase.

The underlying principle of SEC is that particles of different sizeswill elute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size should elute together. Eachsize exclusion column has a range of molecular weights that can beseparated. The exclusion limit defines the molecular weight at the upperend of this range and is where molecules are too large to be trapped inthe stationary phase. The permeation limit defines the molecular weightat the lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

High-performance liquid chromatography (or High pressure liquidchromatography, HPLC) is a form of column chromatography used frequentlyin biochemistry and analytical chemistry to separate, identify, andquantify compounds. HPLC utilizes a column that holds chromatographicpacking material (stationary phase), a pump that moves the mobilephase(s) through the column, and a detector that shows the retentiontimes of the molecules.

Retention time varies depending on the interactions between thestationary phase, the molecules being analyzed, and the solvent(s) used.

V. Pharmaceutical Compositions

It is contemplated that the novel arginases of the present invention canbe administered systemically or locally to inhibit tumor cell growthand, most preferably, to kill cancer cells in cancer patients withlocally advanced or metastatic cancers. They can be administeredintravenously, intrathecally, and/or intraperitoneally. They can beadministered alone or in combination with anti-proliferative drugs. Inone embodiment, they are administered to reduce the cancer load in thepatient prior to surgery or other procedures. Alternatively, they can beadministered after surgery to ensure that any remaining cancer (e.g.cancer that the surgery failed to eliminate) does not survive.

It is not intended that the present invention be limited by theparticular nature of the therapeutic preparation. For example, suchcompositions can be provided in formulations together withphysiologically tolerable liquid, gel or solid carriers, diluents, andexcipients. These therapeutic preparations can be administered tomammals for veterinary use, such as with domestic animals, and clinicaluse in humans in a manner similar to other therapeutic agents. Ingeneral, the dosage required for therapeutic efficacy will varyaccording to the type of use and mode of administration, as well as theparticularized requirements of individual subjects.

Such compositions are typically prepared as liquid solutions orsuspensions, as injectables. Suitable diluents and excipients are, forexample, water, saline, dextrose, glycerol, or the like, andcombinations thereof. In addition, if desired the compositions maycontain minor amounts of auxiliary substances such as wetting oremulsifying agents, stabilizing or pH buffering agents.

Where clinical applications are contemplated, it may be necessary toprepare pharmaceutical compositions-expression vectors, virus stocks,proteins, antibodies and drugs-in a form appropriate for the intendedapplication. Generally, pharmaceutical compositions of the presentinvention comprise an effective amount of one or more arginase variantsor additional agent dissolved or dispersed in a pharmaceuticallyacceptable carrier. The phrases “pharmaceutical or pharmacologicallyacceptable” refers to molecular entities and compositions that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal, such as, for example, a human, asappropriate. The preparation of an pharmaceutical composition thatcontains at least one arginase variant, such as a stabilized multimericarginase or a pegylated arginase isolated by the method disclosedherein, or additional active ingredient will be known to those of skillin the art in light of the present disclosure, as exemplified byRemington's Pharmaceutical Sciences, 18^(th) Ed., 1990, incorporatedherein by reference. Moreover, for animal (e.g., human) administration,it will be understood that preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18^(th) Ed., 1990, incorporated herein by reference). Except insofar asany conventional carrier is incompatible with the active ingredient, itsuse in the pharmaceutical compositions is contemplated.

The present invention may comprise different types of carriers dependingon whether it is to be administered in solid, liquid or aerosol form,and whether it need to be sterile for such routes of administration asinjection. The present invention can be administered intravenously,intradermally, transdermally, intrathecally, intraarterially,intraperitoneally, intranasally, intravaginally, intrarectally,topically, intramuscularly, subcutaneously, mucosally, orally,topically, locally, inhalation (e.g., aerosol inhalation), injection,infusion, continuous infusion, localized perfusion bathing target cellsdirectly, via a catheter, via a lavage, in lipid compositions (e.g.,liposomes), or by other method or any combination of the forgoing aswould be known to one of ordinary skill in the art (see, for example,Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated hereinby reference).

The arginase variants may be formulated into a composition in a freebase, neutral or salt form. Pharmaceutically acceptable salts, includethe acid addition salts, e.g., those formed with the free amino groupsof a proteinaceous composition, or which are formed with inorganic acidssuch as for example, hydrochloric or phosphoric acids, or such organicacids as acetic, oxalic, tartaric or mandelic acid. Salts formed withthe free carboxyl groups can also be derived from inorganic bases suchas for example, sodium, potassium, ammonium, calcium or ferrichydroxides; or such organic bases as isopropylamine, trimethylamine,histidine or procaine. Upon formulation, solutions will be administeredin a manner compatible with the dosage formulation and in such amount asis therapeutically effective. The formulations are easily administeredin a variety of dosage forms such as formulated for parenteraladministrations such as injectable solutions, or aerosols for deliveryto the lungs, or formulated for alimentary administrations such as drugrelease capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a composition contained therein, its use inadministrable composition for use in practicing the methods of thepresent invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include arginasevariants, one or more lipids, and an aqueous solvent. As used herein,the term “lipid” will be defined to include any of a broad range ofsubstances that is characteristically insoluble in water and extractablewith an organic solvent. This broad class of compounds are well known tothose of skill in the art, and as the term “lipid” is used herein, it isnot limited to any particular structure. Examples include compoundswhich contain long-chain aliphatic hydrocarbons and their derivatives. Alipid may be naturally occurring or synthetic (i.e., designed orproduced by man). However, a lipid is usually a biological substance.Biological lipids are well known in the art, and include for example,neutral fats, phospholipids, phosphoglycerides, steroids, terpenes,lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids withether and ester-linked fatty acids and polymerizable lipids, andcombinations thereof. Of course, compounds other than those specificallydescribed herein that are understood by one of skill in the art aslipids are also encompassed by the compositions and methods of thepresent invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the stabilized multimeric or pegylated arginasemay be dispersed in a solution containing a lipid, dissolved with alipid, emulsified with a lipid, mixed with a lipid, combined with alipid, covalently bonded to a lipid, contained as a suspension in alipid, contained or complexed with a micelle or liposome, or otherwiseassociated with a lipid or lipid structure by any means known to thoseof ordinary skill in the art. The dispersion may or may not result inthe formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

VII. Definitions

The term “aa” refers to amino acid(s). Amino acid substitutions areindicated by the amino acid position, e.g. 303, in the molecule using aletter code (the letter in front of the number indicates the amino acidbeing replaced, while the letter after the number indicates the aminoacid being introduced).

As used herein the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

As used herein the terms “protein” and “polypeptide” refer to compoundscomprising amino acids joined via peptide bonds and are usedinterchangeably.

As used herein, the term “fusion protein” refers to a chimeric proteincontaining the protein of interest (i.e., a human arginase or variantthereof) joined (or operably linked) to an exogenous protein fragment(the fusion partner which consists of a non-arginase protein). Thefusion partner may enhance serum half-life, solubility, or both. It mayalso provide an affinity tag (e.g. his-tag) to allow purification of therecombinant fusion protein from the host cell or culture supernatant, orboth.

The terms “in operable combination”, “in operable order” and “operablylinked” refer to the linkage of nucleic acid sequences in such a mannerthat a nucleic acid molecule capable of directing the transcription of agiven gene and/or the synthesis of a desired protein molecule isproduced. The term also refers to the linkage of amino acid sequences insuch a manner so that a functional protein is produced.

The term “K_(m)” as used herein refers to the Michaelis-Menten constantfor an enzyme and is defined as the concentration of the specificsubstrate at which a given enzyme yields one-half its maximum velocityin an enzyme catalyzed reaction.

The term k_(cat) as used herein refers to the turnover number or thenumber of substrate molecule each enzyme site converts to product perunit time, and in which the enzyme is working at maximum efficiency.

The term K_(cat)/K_(m) as used herein is the specificity constant whichis a measure of how efficiently an enzyme converts a substrate intoproduct.

The term “Mn-hArgl” refers to human Arginase I with an Mn (II) cofactor.The term “Co-hArgI” refers to human Arginase I (mutant or native) with aCo (II) cofactor. The term “IC₅₀ ” is the half maximal (50%) inhibitoryconcentration (IC) and thus a measure of effectiveness.

The term “pegylated” refers to conjugation with polyethylene glycol(PEG), which has been widely used as a drug carrier, given its highdegree of biocompatibility and ease of modification. (Harris et al.,2001). Attachment to various drugs, proteins, and liposomes has beenshown to improve residence time and decrease toxicity. (Greenwald etal., 2000; Zalipsky et al., 1997). PEG can be coupled (e.g. covalentlylinked) to active agents through the hydroxyl groups at the ends of thechain and via other chemical methods; however, PEG itself is limited toat most two active agents per molecule. In a different approach,copolymers of PEG and amino acids have been explored as novelbiomaterials which would retain the biocompatibility properties of PEG,but which would have the added advantage of numerous attachment pointsper molecule (providing greater drug loading), and which can besynthetically designed to suit a variety of applications (Nathan et al.,1992; Nathan et al., 1993).

The term “gene” refers to a DNA sequence that comprises control andcoding sequences necessary for the production of a polypeptide orprecursor thereof. The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired enzymatic activity is retained.

The term “subject” refers to animals, including humans.

The term “wild-type” refers to a gene or gene product which has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “variant” or “mutant” refers to a gene or gene productwhich displays modifications in sequence and or functional properties(i.e., altered characteristics) when compared to the wild-type gene orgene product. It is noted that naturally-occurring mutants can beisolated; these are identified by the fact that they have alteredcharacteristics when compared to the wild-type gene or gene product.

VIII. Kits

The present invention provides kits, such as therapeutic kits. Forexample, a kit may comprise one or more pharmaceutical composition asdescribed herein and optionally instructions for their use. Kits mayalso comprise one or more devices for accomplishing administration ofsuch compositions. For example, a subject kit may comprise apharmaceutical composition and catheter for accomplishing directintravenous injection of the composition into a cancerous tumor. Inother embodiments, a subject kit may comprise pre-filled ampoules of astabilized multimeric arginase or isolated pegylated arginase,optionally formulated as a pharmaceutical, or lyophilized, for use witha delivery device.

Kits may comprise a container with a label. Suitable containers include,for example, bottles, vials, and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer may hold a composition which includes an antibody that iseffective for therapeutic or non-therapeutic applications, such asdescribed above. The label on the container may indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above. The kit of the invention willtypically comprise the container described above and one or more othercontainers comprising materials desirable from a commercial and userstandpoint, including buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

IX. EXAMPLES

The following examples serve to illustrate certain preferred embodimentsand aspects of the present invention and are not to be construed aslimiting the scope thereof. In the experimental disclosure whichfollows, the following abbreviations apply: eq (equivalents); M (Molar);μM (micromolar); mM (millimolar); N (Normal); mol (moles); mmol(millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg(milligrams); μg (micrograms); L (liters); ml (milliliters); μl(microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm(nanometers); EC (degrees Centigrade); MW (molecular weight); PBS(phophate buffered saline); min (minutes).

Example 1 Incorporating and Determining Metal Content in Arginase I

Incorporation of Mn²⁺ and Co²⁺ can be achieved by purifying arginase,followed by an incubation step with 10 mM metal at 50° C. for 10minutes. In order to determine the final metal content and identity ofthe arginase preparations, protein samples of Mn-hArgl (145 μM),Co-hArgI (182 μM) and associated dialysis buffers (100 mM Hepes, pH 7.4)were diluted in 2% nitric acid and analyzed by inductively coupledplasma mass spectrometry (ICP-MS, Department of Geological Sciences,University of Texas at Austin) to quantify the protein's cobalt, iron,manganese and zinc content by subtracting the concentration of metalsfound in the dialysis buffer from the metal concentration of the finalprotein samples and dividing by protein concentration. To determineprotein concentrations, an extinction coefficient was calculated forhArgl based on the amino acid sequence (Gill and von Hippel, 1989). Allprotein concentrations for Arginase I were calculated based upon thecalculated ϵ₂₈₀=24,180 M⁻¹ cm⁻¹ in a final buffer concentration of 6 Mguanidinium hydrochloride, 20 mM phosphate buffer, pH 6.5. Forcomparison, arginase concentration was also calculated by BCA assayusing dilutions of BSA as a standard. Using this method it was foundthat arginase samples incubated with Co²+ contain 2.1±0.5 equivalents Coand 0.4±0.1 equivalents Fe, with no detectable amounts of Zn or Mn.Samples incubated with Mn²⁺ contain 1.5±0.2 equivalents Mn and 0.4±0.1equivalents Fe, and no detectable amounts of Zn or Co. Thus, heatincubation is an efficient method for incorporation of cobalt.

Additional studies of cobalt loading have demonstrated that a higherproportion of cobalt loading is achievable and results in a higherspecific activity. The results of these studies is shown on thefollowing table and in FIG. 3.

TABLE 2 Co-Arginase I Cobalt Loading Total Co Total Mn Specific Co TempTime (μg/mg (μg/mg Activity Identity (mM) (° C.) (Min) Arginase)Arginase) (U/mg) APO-Arginase I* NA NA NA <0.025 0.008 24 APO Loading 1*0.1 5 15 0.3 ND 117 Coh-Arg I* 10 20 60 2 0.06  410 APO Loading 2* 1 515 2.4 ND 395 APO Loading 3* 10 20 15 2.8 ND 493 APO Loading 4* 10 20 602.9 ND 489 APO Loading 5 10 37 15 2.8 ND NT APO Loading 6 10 53 15 2.6ND NT Co-ArgI-PEG 10 53 15 3 ND 500 Theoretical 3.4 *Graphed The starreddata are shown in FIG. 3.

Example 2 Cytotoxicty of Co-Arg and its Variants Towards HepatocellularCarcinoma Cells and Metastatic Melanomas

In order to test the in vitro cytotoxicity of engineered arginase,varying concentrations (0-100 nM) of Mn-Argl, Co-ArgI, or Co-hArgIvariants were incubated with HCC (Hep 3b) cells or melanoma (A375) cells(American Type Culture Collection) in 96-well plates at a seedingdensity of 500 cells/well, in DMEM media supplemented with fetal bovineserum. After 24 hours of incubation at 37° C., the cells were treatedwith arginase containing media in triplicate at various concentrations.The control solution was a balanced salt solution in media. The treatedcells were maintained at 37° C. and 5% CO₂. Cells were tested bystandard MTT assay (Sigma-Aldrich) on days 1, 3, 5, & 7 by addition of100 μL/well of MTT (5 mg/mL), and incubated for 4 hours with gentleagitation one to two times per hour. Following this, the solution wasaspirated and 200 μL of DMSO was then added to each well. Absorbance at570 nm was interpreted for each well using an automated plate reader todetermine the relative number of surviving cells compared to controls.The resulting data was fit to an exponential equation to determine anapparent IC₅₀ value for arginase cytotoxicity. The IC₅₀ values from day5 were calculated, yielding an IC₅₀ value for Mn-hArgl of 5±0.3 nM(˜0.18 μg/ml) and a value of 0.33±0.02 nM for Co-hArgl (˜0.012 μg/ml).Thus, the Co-Argl enzyme appears to be 15 fold more cytotoxic than theMn substituted enzyme against HCC. Against the metastatic melanoma cellline (A375) Mn-hArgl resulted in an apparent IC₅₀ of 4.1±0.1 nM (˜0.15μg/m1). Incubation with Co-hArgl lead to a 13-fold increase incytotoxicity with an apparent IC₅₀ of 0.32±0.06 nM (˜0.012 μg/m1).

Example 3 Engineering an Fc-Arginase Fusion Protein for Enhanced In VivoHalf-Life

Fusion to the IgG Fc domain has been employed extensively for prolongingthe in vivo half-lives of therapeutic polypeptides such as the TNF-αinhibitor etanercept (Enbril™). The Fc domain binds to the FcγRnreceptor, which is expressed on vascular endothelium and many othertissues (Roopenian and Akilesh, 2007). The affinity of FcγRn for the IgGFc domain is strongly pH dependent. Binding occurs at the acidic pH ofendosomal compartments allowing the protein to be recycled onto the cellsurface and thus escape proteolytic degradation. At the cell surface,the Fc domain is released from FcγRn because the binding affinity isvery low at physiological pH. Endosomal recycling via FcγRn is estimatedto increase the serum half-life of immunoglobulins at least 4-7 fold, toabout 7-14 days in humans. Fc fusions exploit this property to endowshort lived molecules with a long half-life. However, the human arginaseis a homotrimer and therefore if fused to the IgG Fc, which itself is adimer, the resulting Fc-arginase polypeptide will likely form highmolecular weight aggregates.

This problem was avoided by employing mutant forms of arginase thatdisrupt trimerization and are stable in the monomeric form. Thetrimerization and subunit interface of Arginase I have been studied insome detail (Lavulo et al., 2001). A single amino acid substitution atGlu256Gln has been shown to disrupt trimerization resulting in theformation of monomeric Arginase I enzyme (Sabio et al., 2001). Afterexpression and purification of this variant, the steady-state kineticanalysis revealed nearly identical activity compared to Co-hArgl with ak_(cat)/K_(M) of 1,320 s⁻¹ mM⁻¹.

This construct was then cloned into Fc expression vectors. The Fcexpression vector is a construct based on a pTRC99a plasmid (Amersham)that contains a DsbA leader sequence followed by the IgG Fc codingregion, an EcoRI restriction site and a stop codon. The monomericarginase gene was placed in frame behind the Fc coding region bydigesting both vector and gene with EcoRI, and was subsequently ligatedand transformed into E. coli (BL21) for sequencing and expression. Sincethe IgG Fc is normally a glycosylated protein, expression of recombinantIgGs or of Fc fusions has so far been carried out in recombinantmammalian cells that, unlike bacteria, are capable of N-linkedglycosylation. However, while glycosylation at Asn297 is critical forthe binding to the activating and inhibitory Fcy receptors (FcγRI-III inhumans) it does not have a noticeable effect on the affinity or pHdependent binding to FcγRn (Tao and Morrison, 1989; Simmons et al.,2002). Thus, aglycosylated IgG antibodies expressed in bacteria exhibitserum persistence in primates nearly indistinguishable from that offully glycosylated antibodies expressed in mammalian cells (Simmons etal., 2002). In contrast to prevailing earlier notions, IgG antibodiesand Fc proteins can be expressed efficiently in E. coli up to g/L levelsin fermenters. E. coli expression is technically much simpler andfaster. In addition, since the resulting protein is aglycosylated, itdoes not display glycan heterogeneity, an important issue in theexpression of therapeutic glycoproteins (Jefferis, 2007). The fusionprotein is purified by Protein A chromatography and the yield ofcorrectly folded, dimeric Fc-arginase fusion relative to polypeptidesthat fail to dimerize is quantified by FPLC gel filtrationchromatography. This formulation has led to a highly active and verystable form of human arginase, suitable for in vivo trials.

Example 4

Pegylation of Arginase

Arginase was purified and was then made 10 mM with CoCl₂ and heated at50° C. for 10 minutes. After centrifuging to remove any precipitates,the PEG-5000 arginase was extensively buffer exchanged (PBS with 10%glycerol) using a 100,000 MWCO filtration device (Amicon), andsterilized with a 0.2 micron syringe filter (VWR). All pegylated enzymewas analyzed for lipopolysaccharide (LPS) content using a LimulusAmebocyte Lysate (LAL) kit (Cape Cod Incorporated).

Pegylated Co-hArgl was found to have nearly identical serum stability towild type enzyme and displayed a k_(cat)/K_(M) value of 1690±290 s⁻¹mM⁻¹.

Example 5

Serum Depletion of L-Arg in the Mouse Model

Balb/c mice were treated by single IP injection with 500 μg ofpharmacologically prepared, pegylated Co-hArgl or an equal volume ofPBS. Mice were sacrificed by cardiac veni-puncture for blood collectionat the time points of 0, 48, 72, and 96 hrs. Blood samples wereimmediately mixed 50:50 (v/v) with a 400 mM sodium citrate buffer pH 4,allowed to clot for 30 minutes and centrifuged for serum separation. Theresulting serum was then filtered on a 10,000 MWCO device (Amicon) forthe removal of large proteins and precipitates and the flow-through wascollected for analysis. L-arginine standards, control mouse serum andexperimental samples were derivatized with OPA (Agilent) and separatedon a C18 reverse phase HPLC column (Agilent) (5 μm, 4.6×150 mm)essentially as described by Agilent Technologies (Publication Number:5980-3088) except for modification of the separation protocol slightlyby reducing the flow rate by ½ and doubling the acquisition time to getbetter peak separation. An L-arginine standard curve was constructed byplotting L-Arg peak area versus concentration in order to quantify serumL-Arg levels. A single dose of pharmacologically prepared Co-hArgl wassufficient to keep L-Arg at or below detection limits for over 3 days(FIG. 1).

Example 6

HCC Tumor Xenograft Treatment with Co-hArgl

Nude mice were injected subcutaneously in the flank with ˜10⁶ HCC cellscollected from a 75% confluent tissue culture. After the HCC xenograftedtumors had grown to ˜0.5 cm³ in diameter (Day 9), mice were sorted intotwo groups. The experimental group received a 500 μg IP injection ofpharmacologically optimized Co-hArgl at day 9 and at day 12. The controlgroup received IP injections of PBS at days 9 and 12. As can be seen inFIG. 2, the PBS treated tumors had increased 3-fold in size by day 15.In stark contrast, Co-hArgl treated tumors had decreased in size by day15. Mn-hArgl treated tumors had only been shown to be retarded in growthrate (Cheng et al., 2007). Co-hArgl appears to be a highly effectivechemotherapeutic agent against HCCs both in vitro and in vivo.

Example 7

Disruption of the L-Arginine Balance in the Tumor Microenvironment withCo-hArgl and anti-PD-Ll Ab

Human arginase I (hArgl) is a Mn²⁺ -dependent enzyme that displays lowactivity and low stability in serum. Myeloid-derived suppressor cells(MDSC) express hArgl and nitric-oxide synthase (NOS), which control theavailability of L-arginine in the tumor microenvironment and in turnregulate the function of T-cells. Depletion of L-arginine by MDSC hasbeen correlated to impairment of T-cell anti-tumor function and tumorevasion of host immunity. The expression of enzymes of the L-argininebiosynthetic pathway in peripheral blood mononuclear cells, bone marrowmononuclear cells and CD34⁺ cells was analyzed revealing that thesecells express low levels of OTC and ASS, suggestive of dependence ofthese cells on exogenous/extracellular L-arginine for physiologicalfunction. Based on this finding it is contemplated that long termdepletion of L-arginine may negatively impact the MDSC population andtherefore enhance immune regulation of tumor growth. This hypothesis wastested using engineered hArgI (AEB1102), developed by replacement of theMn²⁺ natural cofactor with Co²⁺ which results in significantly improvedcatalytic activity and serum stability compared to endogenous hArgl. Theengineered enzyme is also pegylated as described above. The effects ofchronic, extensive pegylated Co-hArgl-mediated depletion of L-argininein vivo in the murine CT26 colon-cancer model dosed alone and incombination with anti-PD-L1 and anti-PD-1 monoclonal antibodies (mAbs)were tested.

Female Envigo Balb/c mice (BALB/cAnNHsd) were used in these studies.They were 6-7 weeks old on Day 1 of the test. Test animals wereimplanted subcutaneously on Day 0 with 5.0E+05 CT26.WT cells. All micewere sorted into study groups and treatment was started as follows:

AEB-001-1037

-   -   Group 1: Vehicle (PBS) IP, Q7Dx4; plus Isotype Control, 10 mg/kg        IP, (Q3Dx2; 3off)×4    -   Group 2: AEB1102, (3 mg/kg IP, Q7Dx4)    -   Group 3: anti-PD-L1 Ab, 10 mg/kg IP, (Q3Dx2; 3off)×4    -   Group 4: AEB1102, 3 mg/kg IP, Q7Dx4; plus anti-PD-L1 Ab, 10        mg/kg IP, (Q3Dx2; 3off)×4;

AEB1102 was dosed 4 times, once weekly, (Days 3, 10, 17, 24)

Anti-PD-L1 was dosed 8 times, one on two off, one on three off each week(Days 3, 6, 10, 13, 17, 20, 24, 27)

All animals were observed for clinical signs at least once daily.Individual body weights and tumor volumes were recorded three timesweekly. Individual mice were terminated when tumor size reached a valueof 2000 mm³.

In vivo treatment of CT26 mice with AEB1102 (peglylated Co-hArgl)resulted in a therapeutic effect comparable to standard immunomodulatoryantibodies that target PD-1 and PD-L1. Of significance, combinationtherapy of AEB1102 with anti-PD-1 and PD-L1 mAbs resulted in anapparently synergistic or at least additive anti-tumor effect comparedto AEB1102 alone and immunotherapy alone.

The data from this study is shown graphically in FIG. 4. The datareflect the effect of treatment with a pegylated Co-hArgl in combinationwith an anti-immune checkpoint protein receptor (anti-PD-1) and ligand,(anti-PD-L1). In the figure the upper curve is an isotype controlantibody, the second curve is anti-PD-L1 antibody, the third curve ispegylated Co-hArgl and the lowest curve is the combination treatment ofthe pegylated Co-hArgl and anti-PD-L1 antibody. As seen in the data, thecombination of the 2 agents has a greater than additive effect oninhibition of tumor growth.

The effect on lymphocyte Tcell activation was also measured in samplestaken on day 3. The percentage of total live cells that expressed CD45+in the four groups as well as the percentage of CD45+ cells that werealso CD8+ are shown in Table 3. These data are also shown in graphicalform in FIGS. 6 and 7.

Groups Day 3 CD45+ cells Day 3 CD8+ cells Vehicle 14.8 ± 3.4 19.4 ± 3.9Isotype 13.8 ± 2.2 18.7 ± 2.1 AEB1102 26.5 ± 3.7 18.7 ± 7.1 anti-PD-L113.1 ± 3.5  26.3 ± 3.24 AEB1102 + anti-PD-L1 22.4 ± 2.3 34.2 ± 5.0 Tablereports mean ± SEM

Collectively these results demonstrate that disrupting the L-argininephysiological balance in the tumor microenvironment inhibits tumorgrowth and further sensitizes the tumor to immunotherapy.

Example 8

Effect of treatment of Colon Carcinoma Treatment with Argl and OX40

Suspensions of MC38 colon carcinoma cells were injected into the flanksof female C57BL/6 mice. When tumor volume reached 75-100 mm³ on day 0,mice were randomized into groups. Tumor volume was measured twice a weekusing calipers. Treatments were started on Day 0.

A first group was injected with 10 mg/kg isotype control biweekly for 6weeks, second group was injected with 3 mg/kg co-Arginase I, weekly for6 weeks, a third group was injected with 10 mg/kg anti-OX40ab weekly for6 weeks, and a fourth group was injected with 3 mg/kg co-ArgI and 10mg/kg anti OX40ab weekly for 6 weeks. The data from this study is shownin FIG. 5. Although it appears that the co-Argl was given at asuboptimal dose, a trend is seen in which the combination of ArgI andaOX40 has a more than additive effect on the reduction or inhibition oftumor volume.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods, and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference. Cama et al.,Biochemistry, 42:7748-7758, 2003.

-   Cheng et al., Cancer Lett., 224:67-80, 2005.-   Cheng et al., Cancer Res., 67:309, 2007.-   Cheng et al., Cancer Res., 67:4869-4877, 2007.-   Dillon et al., Med. Sci. Monit., 8:BR248-253, 2002.-   Dowling et al., Cell Mol. Life. Sci., 65(13):2039-55, 2008.-   Durante et al., Clin. Exp. Pharmacol. Physiol., 34:906-911, 2007.-   Ensor et al., Cancer Res., 62:5443-5450, 2002.-   Feun et al., J. Neurooncol., 82:177-181, 2007.-   Gill and von Hippel, Anal. Biochem., 182:319-326, 1989.-   Greenwald et al., Crit. Rev Therap Drug Carrier Syst., 17:101-161,    2000.-   Harkki et al., BioTechnology, 7:596-603, 1989.-   Harris et al., Clin. Pharmacokinet., 40(7):539-51, 2001.-   Hopwood et al., In: Genetic Manipulation of Streptomyces, A    Laboratory Manual, The John-   Innes Foundation, Norwich, Conn., 1985.-   Izzo et al., J. Clin. Oncol., 22:1815-1822, 2004.-   Jefferis, Expert Opin. Biol. Ther., 7:1401-1413, 2007.-   Lavulo et al., J. Biol. Chem., 276:14242-14248, 2001.-   Lopez et al., Febs J., 272:4540-4548, 2005.-   Lordanescu, J. Bacteriol, 12:597 601, 1975.-   Mellor et al., Gene, 24:1-14, 1983.-   Nathan et al., Bioconj Chem., 4:54-62, 1993.-   Nathan et al., Macromolecules, 25:4476-4484, 1992.-   Pardon, Drew, Nature Reviews Cancer 12, 252-264 (April 2012-   Penttila et al., Gene, 61:155-164, 1987.-   Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,    1289-1329, 1990.-   Roopenian and Akilesh, Nat. Rev. Immunol., 7:715-725, 2007.-   Sabio et al., FEBS Lett., 501:161-165, 2001.-   Santhanam et al., Circ. Res., 101:692-702, 2007.-   Savoca et al., Cancer Biochem. Biophys., 7:261-268, 1984.-   Scott et al., Br. J. Cancer, 83:800-810, 2000.-   Shen et al., Cancer Lett., 231:30-35, 2006.-   Sibakov et al., Eur. J. Biochem., 145:567 572, 1984.-   Simmons et al., J. Immunol. Methods, 263:133-147, 2002.-   Tao et al., J. Immunol., 143:2595-2601, 1989.-   Ward, Embo-Alko Workshop on Molecular Biology of Filamentous Fungi,    Helsinki, 119-128, 1989.-   Wheatley and Campbell, Pathol. Oncol. Res., 8:18-25, 2002.-   Yoon et al., Int. J. Cancer, 120:897-905, 2007.-   Zalipsky et al., Bioconjug Chem., 8:111-118, 1997.

1. A method of inhibiting tumor growth in a subject, comprisingadministering a pharmaceutical composition comprising a therapeuticamount of a human Arginase I enzyme comprising a cobalt cofactor and atherapeutic amount of an immuno-oncology agent.
 2. The method of claim1, wherein the tumor comprises arginine auxotrophic tumor cells.
 3. Themethod of claim 1, wherein the human Arginase I enzyme is stabilized byassociation with a stabilizing agent.
 4. The method of claim 3, whereinthe stabilizing agent comprises polyethylene glycol, a synthetic proteinpolymer, an Fc fusion, or albumin.
 5. The method of claim 1, wherein thehuman Arginase I enzyme is pegylated.
 6. The method of claim 1, whereinthe tumor exhibits a reduced or inhibited expression ofargininosuccinate synthetase, ornithine transcarbamylase,argininosuccinate lyase, or a combination thereof.
 7. The method ofclaim 1, wherein the subject is an animal subject.
 8. The method ofclaim 1, wherein the subject is a human cancer patient.
 9. The method ofclaim 1, wherein the immuno-oncology agent enhances the subject's immuneresponse.
 10. The method of claim 9, wherein the immuno-oncology agentinhibits an immune suppressor.
 11. The method of claim 9, wherein theimmuno-oncology agent blocks a checkpoint inhibitor pathway.
 12. Themethod of claim 9, wherein the immuno-oncology agent comprises a PD-1,OX40 or other B7 pathway inhibitor.
 13. The method of claim 11, whereinthe agent is an anti-PD-1 antibody or anti-PD-L1 antibody.
 14. Themethod of claim wherein the immune-oncology agent is an anti-OX40 oranti-OX40L antibody.
 15. The method of claim 13, wherein the agentcomprises pembrolizumab, ipilimumab, atezolizumab or nivolumab.
 16. Themethod of claim 13, wherein the agent comprises ipilimumab.
 17. Themethod of claim 1, wherein the tumor comprises hepatocellular carcinoma,renal cell carcinoma, breast cancer, melanoma, prostate cancer,pancreatic cancer, bladder cancer, colon carcinoma, colorectal cancer,triple negative breast cancer, Hodgkin's lymphoma, gastric cancer,glioblastoma, Merkel cell carcinoma, lung carcinoma, small cell lungcancers or non-small cell lung cancers.
 18. The method of claim 13,wherein the administration of a combination of the human Arginase Ienzyme and the anti-PD-1 antibody, anti-PDL-1 antibody, anti-OX40antibody or anti-OX40L antibody exhibits an additive effect on tumorgrowth inhibition compared to the tumor growth inhibition exhibited byadministering a therapeutic dose of the anti-PD-1 antibody alone or theanti-PDL-1 antibody alone, or the human Arginase I enzyme alone.
 19. Themethod of claim 13, wherein the administration of a combination of thehuman Arginase I enzyme and the anti-PD-1 antibody, the anti-PDL-1antibody, the anti-OX40L antibody or the anti-OX40L antibody exhibits asynergistic effect on tumor growth inhibition compared to the tumorgrowth inhibition exhibited by administering a therapeutic dose of theanti-PD-1 antibody alone, the anti-PD-Li antibody, the anti-OX40antibody or the anti-OX4OL antibody alone or the human Arginase I enzymealone.
 20. The method of claim 13, wherein the human Arginase I enzymeand the anti-PD-1 antibody, the anti-PDL-1 antibody, the anti-OX40antibody or the anti-OX4OL antibody are administered concurrently. 21.The method of claim 13, wherein the human Arginase I enzyme and theanti-PD-1 antibody, the anti-PDL-1 antibody, the anti-OX40 antibody orthe anti-OX4OL antibody are administered sequentially.
 22. The method ofclaim 1, wherein the human Arginase I enzyme displays a k_(cat)/K_(M)for the hydrolysis of arginine between 400 mM⁻¹ s⁻¹ and 4,000 mM⁻¹ s⁻¹at pH 7.4 and 37° C.
 23. The method of claim 1, wherein the humanArginase I enzyme comprises a ratio of cobalt to arginase of from 2 to 3μgCo/mg arginase.
 24. The method of claim 1, wherein the human ArginaseI enzyme is produced by contacting an arginase apoenzyme with cobalt ora cobalt ion at a temperature of from 30° C. to 55° C. for a period offrom 15 minutes to 60 minutes.
 25. A method of treating cancer in acancer patient comprising administering to said patient a therapeuticamount of a pharmaceutical composition comprising a pegylated humanArginase I enzyme comprising a cobalt cofactor and an immune systemmodulating therapy comprising administering a pharmaceutical compositioncomprising an immuno-oncology agent.
 26. The method of claim 25, whereinthe pharmaceutical composition comprising a pegylated human Arginase Ienzyme comprising a cobalt cofactor and a pharmaceutical compositioncomprising an immuno-oncology agent are administered concurrently. 27.The method of claim 25, wherein the pharmaceutical compositioncomprising a human Arginase I enzyme comprising a cobalt cofactor and apharmaceutical composition comprising an immuno-oncology agent areadministered sequentially.
 28. The method of claim 25, wherein atherapeutic amount of the pegylated human Arginase I enzyme comprising acobalt cofactor is from about 0.01 mg/kg to about 7.5 mg/kg.
 29. Themethod of claim 25, wherein a therapeutic amount of the pegylated humanArginase I enzyme comprising a cobalt cofactor is from about 0.05 mg/kgto about 5 mg/kg.
 30. The method of claim 25, wherein a therapeuticamount of the pegylated human Arginase I enzyme comprising a cobaltcofactor is from about 0.1 mg/kg to about 5 mg/kg.
 31. The method ofclaim 25, wherein the immuno-oncology agent is an anti-PD-1 antibody, ananti-PD-L1 antibody, an anti-OX40 antibody or an anti-OX40L antibody.32. The method of claim 31, wherein the immuno-oncology agent isselected from pembrolizumab, ipilimumab, atezolizumab and nivolumab. 33.The method of claim 31, wherein the cancer patient is treated forhepatocellular carcinoma, renal cell carcinoma, breast cancer, melanoma,prostate cancer, pancreatic cancer, bladder cancer, colon carcinoma,colorectal cancer, triple negative breast cancer, Hodgkin's lymphoma,gastric cancer, glioblastoma, Merkel cell carcinoma, lung carcinoma,small cell lung cancer or non-small cell lung cancer.
 34. The method ofclaim 25, wherein the pharmaceutical composition comprising a pegylatedhuman Arginase I enzyme comprising a cobalt cofactor is administeredparenterally.
 36. The method of claim 25, wherein the pharmaceuticalcomposition comprising a pegylated human Arginase I enzyme comprising acobalt cofactor is administered topically, intravenously, intradermally,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostaticaly, intrapleurally, intratracheally,intraocularly, intranasally, intravitreally, intravaginally,intrarectally, intramuscularly, subcutaneously, subconjunctival,intravesicularlly, mucosally, intrapericardially, intraumbilically,orally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, via acatheter, or via a lavage.
 37. The method of claim 25 wherein thepharmaceutical composition is administered intravenously.
 38. A methodof treating cancer in a cancer patient comprising administering to saidpatient an arginine depleting agent and a checkpoint pathway inhibitor.39. The method of claim 38, wherein the therapeutic effect of treatmentwith said arginine depleting agent and a checkpoint pathway inhibitor isadditive as compared to treatment the arginine depleting agent alone orsaid checkpoint pathway inhibitor alone.
 40. The method of claim 38,wherein the therapeutic effect of treatment with said arginine depletingagent and a checkpoint pathway inhibitor is synergistic as compared totreatment the arginine depleting agent alone or said checkpoint pathwayinhibitor alone.
 41. The method of claim 38, wherein the treatmentresults in from 50% to 99% reduction in serum arginine in the patient.42. The method of claim 38, wherein the treatment results in from 90% to99% reduction of serum arginine in the patient.
 43. The method of claim38, wherein the treatment results in reduction of serum arginine in thepatient to an undetectable level.
 44. The method of claim 38, whereinthe arginine depleting agent comprises an arginase enzyme, an argininedeiminase enzyme or a combination thereof.
 45. The method of claim 38,wherein the enzyme is a human enzyme.
 46. The method of claim 38,wherein said enzyme is an engineered human arginase.
 47. The method ofclaim 38, in which said enzyme is stabilized by conjugation orassociation with an Fc fragment, pegylation, albumin or a syntheticprotein polymer.
 48. A method of inhibiting tumor growth in a subject,comprising administering a pharmaceutical composition comprising atherapeutic amount of a human arginase I enzyme or a mycoplasma argininedeiminase enzyme and a therapeutic amount of an immuno-oncology agent.